Abstract
Endoglin (ENG), a co-receptor for several TGFβ-family cytokines, is expressed in dividing endothelial cells alongside ALK1, the ACVRL1 gene product. ENG and ACVRL1 are both required for angiogenesis and mutations in either gene are associated with Hereditary Hemorrhagic Telangectasia, a rare genetic vascular disorder. ENG and ALK1 function in the same genetic pathway but the relative contribution of TGFβ and BMP9 to SMAD1/5/8 activation and the requirement of ENG as a co-mediator of SMAD phosphorylation in endothelial cells remain debated. Here, we show that BMP9 and TGFβ1 induce distinct SMAD phosphorylation responses in primary human endothelial cells and that, unlike BMP9, TGFβ only induces SMAD1/5/8 phosphorylation in a subset of immortalized mouse endothelial cell lines, but not in primary human endothelial cells. We also demonstrate, using siRNA depletion of ENG and novel anti-ENG antibodies, that ENG is required for BMP9/pSMAD1 signaling in all human and mouse endothelial cells tested. Finally, anti-ENG antibodies that interfere with BMP9/pSMAD1 signaling, but not with TGFβ1/pSMAD3 signaling, also decrease in vitro HUVEC endothelial tube formation and inhibit BMP9 binding to recombinant ENG in vitro. Our data demonstrate that BMP9 signaling inhibition is a key and previously unreported mechanism of action of TRC105, an anti-angiogenic anti-Endoglin antibody currently evaluated in clinical trials.
Highlights
Endoglin (ENG), a dimeric membrane glycoprotein known as CD105, is differentially expressed at the surface of hypoxic or dividing endothelial cells (EC) and is expressed in the endothelium of virtually all solid tumors [1,2]
To understand the role of ENG in endothelial cell signaling, we first tested the ability of TGFb1 and BMP9 to induce SMAD phosphorylation in primary human ECs
Using quantitative PCR, we confirmed the up-regulation of the SMAD6 mRNA upon BMP9 stimulation and of the SERPINE1/PAI-1 mRNA upon TGFb1 stimulation of HUVECs (Fig. S1), genes previously reported to be strongly induced in endothelial cells by BMP9 [12] or TGFb [15], respectively
Summary
Endoglin (ENG), a dimeric membrane glycoprotein known as CD105, is differentially expressed at the surface of hypoxic or dividing endothelial cells (EC) and is expressed in the endothelium of virtually all solid tumors [1,2]. ENG can associate with a number of Type I and Type II TGFb receptors and is defined as a Type III TGFb co-receptor based on its homology to TGFBR3 (a.k.a. betaglycan) and its ability to bind diverse TGFb-family cytokines including Activin, TGFb1, BMP2, BMP7 and BMP9/ 10, in vitro [3] or in cell culture assays [4]. Most individuals affected with Hereditary Hemorrhagic Telangectasia (HHT), an autosomal-dominant genetic disorder leading to severe vascular malformations, carry heterozygous mutations in either ENG (HHT type 1) or ACVRL1 (HHT type 2) [7]. Homozygous Eng and Alk mutant mouse embryos closely phenocopy each other and die at day 11.5 from severe vascular malformations attributed to defective angiogenesis [8]. ALK1 binds to ENG [9]
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